Material and methods - On the significance of song amplitude in birds

Study species and site

Chaffinches have a small repertoire of 1–6 song types, with the majority of birds having 2 or 3 (Slater 1983). Birds that sing more than one song type typically sing short sequences of one song type and then switch to another type (Riebel and Slater 1999b; Brumm and Slater 2006a). As in most songbirds, the 2 major functions of chaffinch song are mate attraction and territory defense, and therefore variations in song characteristics are meaningful to both female and male receivers (Slater 1981; Riebel and Slater 1998; Leitão et al. 2006). In central Europe, male chaffinches establish their territories from mid-February until March, and egg laying does not typically begin until mid-April (Bauer et al. 2005). The song level measurements and playback experiments were carried out in Starnberg district, Germany.

Study sites were selected based on a good visibility of the subjects and away from noise sources, such as busy roads or railway lines.

Song level measurements

We measured the SPLs of the songs from 20 male chaffinches between 19 and 23 March 2010 from 0700 to 1100 h with a CEL 573.B1 Sound Level Analyser. The Sound Level Analyzer was used in a measurement mode as a precision (class 1) real time sound level meter, which allowed us to manually store measurements in an internal memory. For an average of 12

songs per male (range 10–21 songs), we recorded the A-weighted SPL with an integration time of 125 ms (dB SPL, re. 20 lPa).

Quantifying song amplitude in the field is challenging because several factors may affect the measurements. First, birds may adjust their song amplitude in response to varying levels of background noise (termed the Lombard effect, reviewed in Brumm and Slabbekoorn 2005) and dependent on the social context, that is, the presence and distance of targeted receivers (Brumm and Todt 2004; Cynx and Gell 2004; Brumm and Slater 2006b). Second, the measuring procedure is also crucial because the measured sound pressure values vary with distance from the singing bird as well as its orientation (Brumm 2004b). Finally, the environmental acoustics of the habitat can affect sound level measurements due to varying levels of sound absorption by differences in vegetation as well as air temperature and humidity (Wiley and Richards 1982). The effect of the latter increases with the recording distance; thus, it can be minimized by keeping the recording distance to a minimum with no obstacles in the direct sound path between the measuring microphone and the singing bird.

We did our measurements (and the subsequent playback experiments) during a short period of just a few early days in the breeding season, which had 2 advantages: all subjects were in the same breeding stage (i.e., territory establishment, several weeks before egg laying) and there was no foliage, which allowed good visibility of the birds and minimal sound absorption and scattering (Blumenrath and Dabelsteen 2004). Song levels were only recorded provided there were no obstacles (e.g., branches) between the singing bird and the sound level meter, and the bird was closer than 30 m (mean 17.5 m, range 10–27 m, assessed with a Leica Rangemaster 800 CRF laser range finder). Readings were only taken from an angle of incidence of about 90° in relation to the animal’s longitudinal axis, and the microphone of the sound level meter pointing directly at the singing bird. For each song, 2 measurements were taken: one of the maximum SPL of the song and one immediately after the end of the song (see above). The second reading was used as a proxy for the environmental noise during the song (see below). Air temperature and humidity were measured with a Conrad WS-7138 thermo/hygrometer. To ensure that all birds were in a similar social context, we only measured individuals that had their territory within earshot of other singing chaffinches. To further control for a potential effect of the density and distance of neighboring males, we recorded the number of males singing within earshot as well as the distance from the measured bird to the nearest singing neighbor.

Playback experiment

Construction of playback stimuli

Each male was tested with the songs of a different source male. All source males were recorded in previous years more than 1300 km away from the test sites (Brumm et al. 2009a), which ensured that birds tested were neither familiar with the songs nor the singer. This is advantageous because chaffinches have been found to show stronger territorial responses toward strange than toward familiar songs (Slater 1981). From the recording of each source male, we took one high-quality recording of each of 2 song types. These 2 songs were then high-pass filtered at 1.4 kHz and subsequently normalized to 95%. Eight copies of one song type followed by 5 copies of the other were then copied in regular intervals into a 2-min playback file to form a song sequence with a song rate of 6.5 songs per minute. This song rate, and the switch in song types after 8 renditions, was derived from the average song performance values for chaffinches measured in a sample of over 100 individuals (Brumm et al. 2009a). All digital editing and construction of the playback files were done with Avisoft SASLab Pro (Raimund Specht, Berlin). Finally, the playback files were stored in the digital memory of a playback device (Foxpro Scorpion model X1-A) that allowed a remote controlled broadcast of the files. Half of the stimulus files were randomly assigned to the high-amplitude treatment, the other half to be low-amplitude treatment. The volume control of the playback device was set to values that resulted in 87 dB SPL at 1 m distance for the high-amplitude songs and 78 dB SPL at 1 m distance for the low-amplitude playback (as measured with a CEL 573.B1 Sound Level Analyser). These SPL values were consistent with the high and low end of the distribution of chaffinch song SPL measured before the playback (see below). The 2 experimental treatments were carried out alternating, that is, the first subject received one treatment, the second the other, and so on.

Procedure

Playback experiments were carried out from 24 to 31 March 2010 between 0630 and 0930 h.

As with the song level measurements, we restricted the playback experiment to a brief period of a few days earlier in the breeding season to ensure that all males were in a similar breeding stage, that is, territory establishment and pair formation. Females were only observed in some of the territories on the last 3 days of the experimental period.

In total, we tested 22 males, 11 with the high-amplitude treatment and 11 with the low-amplitude treatment. At the beginning of each experiment, each bird was observed until at

least 2 song posts could be mapped. Then a dummy bird (a taxidermic mount of a male chaffinch that was stuffed in a singing posture) was mounted on a tripod in 170 cm height immediately above the playback device. The tripod with the dummy and the playback device was positioned in between 2 song posts of the targeted bird to ensure that all playbacks were done within the birds’ territories. (Although the song playback was fully replicated, the use of only one dummy could be regarded as pseudoreplication. However, the same taxidermic mount was used in all experiments so that any potential bias induced by the particular dummy used was the same for high- and low-amplitude playbacks.).

Previous playback studies with chaffinches found that the latency to approach the loudspeaker, the time spent within 5 m of it, the closest approach, the number of flights, and the number of calls are the most useful response variables (Slater 1981; Slater and Catchpole 1990; Leitão and Riebel 2003). We adopted these response variables, and in addition also considered the number of songs because chaffinches decrease their song output in response to playbacks inside their territories (Slater 1981).

The males’ territorial behaviors were registered 2 min before, during, and after the playback. During these periods, we recorded their vocalizations with a Sennheiser Me 66 directional microphone connected to a Marantz PMD 660 solid state recorder. The recorded digital audio files had a sampling rate of 44.1 kHz and an accuracy of 16 bit. A spoken commentary reporting the other response variables was simultaneously recorded with the same equipment. The playback was started after the 2-min preplayback observation period and when the bird was not further away than 30 m from the dummy bird (measured with a Leica Rangemaster 800 CRF laser range finder). The initial distance between the subjects and the dummy at the beginning of the playback did not differ systematically between the 2 treatments (Welch 2-sample t-test, t = 1.06, df = 18.20, P = 0.30; mean distance high-amplitude group: 23.7 m [range 15–30 m] and low-high-amplitude group: 21.6 m [range 13–29 m]) nor did the time of day when the animals were tested (t = 0.70, df = 18.71, P = 0.49).

Data analysis

The reading of the sound level meter gives the SPL of all incoming sound energy, that is, the song and the background noise. To calculate the SPL of the song without the background noise, we subtracted the second SPL value taken immediately after each song from the SPL measurement taken during the song (for details of the logarithmic calculation procedure, see Brumm et al. 2009b). However, all birds were recorded in areas with low levels of ambient noise and because the measurements were done very close to the birds, the background noise

had very little or virtually no effect on measured values of song (signal-to-noise ratios: 10–26 dB). In a second step, the SPL values for each song were normalized to the standard distance of 1 m according to the spherical spreading of sound. The SPL in 1 m distance is L1m = 6 log(d) + Ld, where d is the distance (in m) from which the SPL measurement was taken, and Ld is the SPL in distance d. Finally, the mean L1m value for all the songs of each male were calculated and used for further analysis.

The validity of our method was checked by calculating a repeatability score. Therefore, we compared SPL values within 14 males that have been taken at different distances. For each individual, the mean SPL values of the closest and the furthest recording were correlated with each other. A strong correlation would show that variation within males is smaller than the variation between males, indicating consistent amplitude differences between males and high internal validity of the method.

To control for potential environmental effects on the song amplitude measurements, we conducted a general linear model with time of the day, temperature, humidity, number of singing neighbors, distance to the nearest neighbor, and background SPL as explanatory variables. Nonsignificant variables were backward eliminated.

When analyzing the data of the playback experiments, pairwise comparisons between the response variables revealed that all but 2 (number of calls uttered during the playback and the latency of approach) were correlated with other variables. Therefore, to obtain uncorrelated response measures, we performed a principal component analysis (PCA) on the correlated variables (during the playback: number of songs and flights, closest approach, time spent within 5 m; after the playback: number of songs, calls and flights and time spent within 5 m during the postplayback period). A Kaiser–Meyer–Olkin test confirmed that our correlation matrix was suitable for a PCA (sampling adequacy = 0.65). The PCA yielded 2 components with an eigenvalue higher than 1, which accounted for 70.3% of the overall variance. PC1 was mainly a measure of number of flights and songs during the playback, of closest approach and of number of songs after the playback, whereas the other variables loaded to similar extent on the 2 main components (Table 1).

Comparisons of the PCA scores, as well as comparisons of the raw response variables, were made with Welch 2-sample t-tests, following the recommendations of Ruxton (2006).

For nonnormally distributed data, we used ranks instead of the original ratio measurements.

All statistical tests were performed with R 2.8.1 (R Development Core Team 2008) and SPSS 15.0 (SPSS Inc.). All tests were 2-tailed.

Results

We found considerable differences in the mean song amplitude between males, with a maximum between-individual difference of 9 dB (range between males: 78–87 dB, mean value of all males 82 dB; Fig. 4). Our method of measuring song amplitude in the field proved to be highly repeatable with a repeatability score of r² = 0.81. None of the environmental variables varied significantly with the measured SPLs. The only exception being air temperature (F = 6.11, df = 1.19, P = 0.02): song amplitude tended to be higher when temperature was low. However, this effect was fairly weak, with temperature explaining less than 20% of the variation in song amplitude (r² = 0.19).

Figure 4. Song amplitudes of 20 free-ranging male chaffinches measured in their natural habitat. Mean and standard error are given for each individual.

Before the playback, none of the measured behavioral variables differed significantly between the subjects that received the high-amplitude playback and those that received the low-amplitude playback (number of songs [Welch 2-sample t-test: t = 0.40, df = 16.14, P = 0.69], number of calls [t = 1, df = 10, P = 0.34] and number of flights [t = 21.17, df = 19.37, P = 0.26]).

During the playback, all 22 males approached the loudspeaker, and there was no significant difference in the latency of approach during high- and low-amplitude playback (t

= 0.21, df = 19.15, P = 0.83; Fig. 5). This suggests that the playback amplitude had no effect on the detectability of the simulated intruder.

PCA revealed that males responded significantly stronger in response to high-amplitude than to low-amplitude songs according to PC1 (t = 2.79, df = 14.59, P = 0.01) but not

according to PC2 (t = 1.45, df = 11.67, P = 0.17). PC1 explained more than twice as much variation in the data than PC2. To further analyze the response difference between the 2 experimental groups, we investigated each response variable singly (Fig. 5): During playback, birds exposed to loud songs sang less (t = 22.59, df = 16.76, P = 0.02), spent more time within 5 m of the loudspeaker (t = 5.62, df = 16.20, P < 0.01), and approached closer (t = 24.38, df = 17.65, P < 0.01) than those exposed to low-amplitude song. The number of calls (t = 1.68, df = 20.00, P < 0.11) and the number of flights (t = 1.85, df = 18.19, P = 0.08) did not differ significantly between the treatment groups. During the postplayback period, birds also spent more time within 5 m of the loudspeaker when exposed to loud song (t = 4.01, df = 10, P <

0.01), while the number of songs (t = 20.93, df = 17.12, P = 0.37) and calls (t = 1.01, df = 18.15, P = 0.32) and the number of flights (t = 0.30, df = 19.38, P = 0.77) did not differ significantly. All significant differences between the single response variables were retained after Bonferroni-Holm correction.

Table 1. PCA including 8 response variables

PC1 PC2

during the playback

number of flights 0.83 -0.19 number of songs -0.85 0.08

time within 5m 0.54 0.75

closest approach -0.82 -0.19 after the playback

number of flights 0.52 -0.57 number of songs -0.75 0.22 number of calls 0.44 -0.62

time within 5m 0.67 0.59

All principal components with an eigenvalue higher than 1 are given (PC1 and PC2). Loadings of variables that made an important contribution to the PC score (<0.5) are indicated in bold.

Discussion

We found considerable differences in song amplitude between male chaffinches, with the loudest male in our sample of 20 birds singing on average 9 dB SPL higher than the male with the softest songs. Similar maxima between individual differences in song amplitude have also been reported in previous studies on free-ranging songbirds (Dabelsteen 1981; Brumm 2009).

Our playback experiment demonstrated that this 9 dB difference in song amplitude significantly affected the territorial behavior of male chaffinches: During a simulated territorial intrusion, males reacted more strongly in response to high-amplitude songs than to low-amplitude songs. In particular, males sang less and approached the dummy closer when we broadcasted high-amplitude songs, and they stayed longer in its vicinity, both during and after the playback.

Figure 5. Responses of territorial chaffinches to playback of loud and soft songs, across 10 raw response variables. X-axis shows the playback amplitude (high: 87 dB SPL at 1 m distance, low: 78 dB SPL at 1 m distance). Medians and interquartile ranges are shown for N = 11 males for each treatment. Statistically significant differences between the 2 playback treatments were found for the number of songs, the time within 5 m, and the minimum approach distance during the playback and the time within 5 m after the playback (see text).

The differences in response strength cannot be explained by a limited audibility of the low-amplitude songs because there was no significant difference in the latency with which the tested birds approached the low- and high-amplitude playback. The validity of our results is further confirmed by a comparison with earlier playback studies with chaffinches (Slater 1981; Slater and Catchpole 1990; Leitão and Riebel 2003). Territorial males in these studies showed similar responses to playback in terms of number of songs during the playback, time spent within 5 m of the loudspeaker, and nearest approach as the males in our study did.

Moreover, as we presented a stuffed male chaffinch together with the song stimulus and because all birds closely approached the dummy, we can exclude that territory owners simply perceived the simulated loud singers to be spatially closer. In one case, a territory owner showed close approach and antagonistic behavior toward the dummy before the playback had

even started, which highlights the fact that the birds recognized the taxidermic mount as a conspecific male and potential rival.

Modulation of territorial behavior

The findings of our playback experiment indicate that chaffinches exhibit stronger territorial behaviors toward rival males singing loudly than toward rivals that produce softer songs. On a proximate level, louder songs can be interpreted as stronger stimuli, with more sound energy stimulating the listener’s sensory apparatus and thus leading to an increased arousal and stronger responses. Such a proximate mechanism may be sufficient to account for the differential reaction in our 2 treatment groups. However, as territory defense is costly (Davies 1980; Huntingford and Turner 1987), it will be adaptive for territory holders to adjust the strength of response toward an intruder to the level of threat the intruder poses (de Kort et al.

2009a).

At least 3 mutually nonexclusive explanations may account for why rival males with loud songs are a stronger threat to territory owners: 1) loud singers are presumably highly motivated and thus more likely to escalate a territorial interaction. Several studies have demonstrated such a correlation between vocal amplitude and motivation: male nightingales increase their song amplitude when countersinging with conspecific males (Brumm and Todt 2004). Juvenile tree swallows Tachycineta bicolor and barn swallows Hirundo rustica increase the amplitude of their begging calls with increasing hunger (Leonard and Horn 2001;

Boncoraglio and Saino 2008). So, if high-amplitude songs signal motivation to fight, then territory holders need to put more effort into defending their territory against a loud intruder.

2) Loud singers might be physically stronger than soft singers and thus pose a bigger threat to territory owners. However, body size, a physical attribute which may be of importance in intrasexual aggression, has been found to be unrelated to song amplitude in birds (Brumm 2009). 3) Loud intruders may be more likely to win extrapair copulations from the territorial female and thus pose a higher risk of loosing paternity to territory owners. This seems plausible as loud intruders will be detected over greater distances and as female songbirds are known to prefer loud songs (Searcy 1996; Ritschard et al. 2010). However, in the present case, this explanation cannot explain the modulation of territorial behavior because almost all the birds tested were not mated at the time of the experiment.

Studies on frogs indicate that a male’s response to calls of different amplitude is affected by the density and distance of neighboring males (e.g. Rose and Brenowitz 1991; Burgmeister et al. 1999). Marshall et al. (2003) found that the stimulus amplitude at which male spring

peepers Pseudacris crucifer respond aggressively is positively correlated with the amplitude of the advertisement call of its nearest neighbor. In other words, the closer the nearest calling neighbor, the higher the aggressive threshold of a male. By contrast, we found no effect of the distance to the nearest neighbor or the number of neighbors on the response strength of territorial chaffinches. During our experiments, the distance to the nearest singing neighbor varied markedly between individuals (30–190 m), but the overall number of neighbors was similar between all subjects (all birds had 1 or 2 neighbors). Although frog choruses, in which numerous males call simultaneously in big aggregations, are rather different from the more

Im Dokument On the significance of song amplitude in birds – function, mechanisms, and ontogeny (Seite 32-43)